2.1 Aging
It is estimated that in the next 25 years, the number of individuals over the age of 65 in the United States will double (U.S. Department of Health and Human Services, 2003). With increased chronological age, there is progressive attrition of homeostatic reserve of all organ systems (Resnick and Dosa, 2004). As a consequence, aged individuals have a dramatically increased risk of numerous debilitating diseases including bone fractures, cardiovascular disease, cognitive impairment, diabetes and cancer (Resnick and Dosa, 2004). Therefore, as American demographics shift, increasing demands are placed on our health care system (Crippen, 2000). Identifying strategies to prevent or delay age-associated frailty and diseases is imperative for maintaining the health of our population as well as our nation's economy.
The molecular basis of the progressive loss of homeostatic reserve with aging is controversial (Kirkwood, 2005b; Resnick and Dosa, 2004). There is strong evidence that genetics contribute significantly to lifespan and end-of-life fitness (Hekimi and Guarente, 2003). This was demonstrated by identifying single genes that when mutated or overexpressed attenuate and extend lifespan, respectively (Kurosu et al., 2005). Many of the genes that regulate lifespan affect the growth hormone (GH)/insulin-like growth factor 1 (IGF1) axis, which controls cellular proliferation and growth (Kenyon, 2005). Suppression of this axis extends lifespan significantly and delays age-related diseases (Bartke, 2005).
Alternatively, the disposable soma theory of aging posits that aging is the consequence of accumulation of stochastic molecular and cellular damage (Kirkwood, 2005b). The precise nature of the damage that is responsible for aging-related degenerative changes remains ill-defined, but may include mitochondrial damage, telomere attrition, nuclear dysmorphology, accumulation of genetic mutations, DNA, protein or membrane damage.
There are several lines of evidence to support the notion that DNA damage is one type of molecular damage that contributes to aging. At the forefront of this is the observation that the majority of human progerias (or syndromes of accelerated aging) are caused by inherited mutations in genes required for genome maintenance, including Werner syndrome, Cockayne syndrome, trichothiodystrophy and ataxia telangiectasia (Hasty et al., 2003). Furthermore both DNA lesions (Hamilton et al., 2001) and genetic mutations caused by DNA damage (Dolle et al., 2002) accumulate in tissues with aging. Finally, mice harboring germ-line mutations that confer resistance to genotoxic stress are long-lived (Maier et al., 2004; Migliaccio et al., 1999).
ERCC1-XPF is a highly conserved structure-specific endonuclease that is required for at least two DNA repair mechanisms in mammalian cells: nucleotide excision repair (Sijbers et al., 1996) and DNA interstrand crosslink repair (Niedernhofer et al., 2004). Genetic deletion of either Ercc1 or Xpf in the mouse causes an identical and very severe phenotype (McWhir et al., 1993; Tian et al., 2004; Weeda et al., 1997). Embryonic development of null mice is normal, but postnatally they develop numerous symptoms associated with advanced age including epidermal atrophy and hyperpigmentation, visual impairment, cerebral atrophy with cognitive deficits, cerebellar degeneration, hypertension, renal insufficiency, decreased liver function, anemia and bone marrow degeneration, osteoporosis, sarcopenia, cachexia, and decreased lifespan (Niedernhofer et al., 2006; Prasher et al., 2005; Weeda et al., 1997, and see International Patent Application Publication No. WO2006/052136).
To determine if this progeroid phenotype had commonalities with the natural aging process, the transcriptome from the liver of Ercc1−/− mice was compared to that of old wild type mice and a highly significant correlation was identified (Niedernhofer et al., 2006). Similar expression changes were also identified in young wild type mice after chronic exposure to a DNA damaging agent. This provides direct experimental evidence that DNA damage induces changes that mimic aging at the fundamental level of gene expression.
Gene ontology classification of the expression data was used to predict pathophysiologic changes that were similar in Ercc1−/− mice and old wild type mice (Niedernhofer et al., 2006). These predictions were tested comparing Ercc1−/− mice, young and old wild type mice. For all predictions tested, Ercc1−/− were more similar to old mice than to their wild type littermates despite the vast difference in age (3 weeks vs. 120 weeks). Both Ercc1−/− and old mice displayed hyposomatotropism, hepatic accumulation of glycogen and triglycerides, decreased bone density, increased peroxisome biogenesis, increased apoptosis and decreased cellular proliferation. Therefore, Ercc1−/− and old mice share not only broad changes in gene expression, but also endocrine, metabolic and cell signaling changes. This implies that ERCC1-deficient mice are an accurate and rapid model system for studying systemic aging in mammals. A case of human progeria caused by ERCC1-XPF deficiency with symptoms near-identical to those observed in ERCC1-deficient mice has been reported (Niedernhofer et al., 2006). Therefore function of ERCC1-XPF is conserved from man to mouse and the discovery of what is driving aging-like degenerative changes in ERCC1-deficient mice will have direct implications for human health.
A number of the degenerative changes associated with normal aging may be manifested in an accelerated form and/or in younger individuals. Examples of such degenerative disorders include neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, Parkinson's disease and osteoporosis, and joint degenerative conditions such as osteoarthritis, rheumatoid arthritis and intervertebral disc degeneration.
2.2 Free Radicals, Aging and Degeneration
Cells undergo some degree of oxidative stress by way of generating reactive oxygen species (“ROS”) and reactive nitrogen species (“RNS”). Specifically, the cellular respiration pathway generates ROS and RNS within the mitochondrial membrane of the cell (Kelso et al., 2001). Reactive oxygen species include free radicals, reactive anions containing oxygen atoms, and molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Specific examples include superoxide anion, hydroxyl radical, and hydroperoxides.
Naturally occurring enzymes, such as superoxide dismutase (“SOD”) and catalase, detoxify ROS and RNS radicals to allow normal metabolic activity to occur. Significant deviations from cell homeostasis, such as hemorrhagic shock, lead to an oxidative stress state, thereby causing “electron leakage” from the mitochondrial membrane. This “electron leakage” produces an excess amount of ROS for which the cell's natural antioxidants cannot compensate. Specifically, SOD cannot accommodate the excess production of ROS associated with hemorrhagic shock which ultimately leads to premature mitochondria dysfunction and cell death via apoptosis (Kentner et al., 2002).
Cardiolipin (“CL”) is an anionic phospholipid exclusively found in the inner mitochondrial membrane of eukaryotic cells (Iverson and Orrenius, 2002). Under normal conditions, the pro-apoptotic protein cytochrome C is anchored to the mitochondrial inner membrane by binding with CL (Tuominen, et al., 2002). The acyl moieties of CL are susceptible to peroxidation by reactive oxygen species. When ROS are generated within mitochondria in excess quantities, cytochrome C bound to CL can function as an oxidase and induces extensive peroxidation of CL in the mitochondrial membrane (Kagan et al., 2005a and 2005b).
The peroxidation of the CL weakens the binding between the CL and cytochrome C (Shidoji, et al., 1999). This leads to the release of the cytochrome C into the mitochondrial intermembrane space, inducing apoptotic cell death. Further, the peroxidation of CL has the effect of opening the mitochondrial permeability transition pore (“MPTP”; Dolder et al., 2001; Imai et al., 2003). Accordingly, the mitochondrial membrane swells and releases the cytochrome C into the cytosol. Excess cytochrome C in the cytosol leads to cellular apoptosis (Iverson et al., 2003).
Moreover, mitochondrial dysfunction and cell death may ultimately lead to multiple organ failure despite resuscitative efforts or supplemental oxygen supply (Cairns, 2001). Reduction of oxidative stress delays, even inhibits, physiological conditions that otherwise might occur, such as hypoxia.
One of the limitations of SOD is that it cannot easily penetrate the cell membrane. However, nitroxide radicals, such as TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) and its derivatives, have been shown to penetrate the cell membrane better than SOD and inhibit the formation of ROS, particularly superoxide, due to their reduction by the mitochondrial electron transport chain to hydroxyl amine radical scavengers (Wipf et al., 2005a).
Examples of antioxidant agents include agents set forth in US 2007161544 and US2007161573, such as, for example, XJB-5-131.
The aging-related and degenerative changes described above are associated with deterioration in the context of impaired regenerative capacity. There appears to be an inverse relationship between the maximum lifespan of a species and the amount of ROS and RNS that species produces (Finkel, 2000). Caloric restriction, which reduces ROS and RNS production, promotes longevity and delays the onset of age-related diseases (Heilbronn, 2003). Thus, effective ROS and RNS scavengers are potential therapeutic agents for age-related pathologies and degenerative conditions.